The Molecular Mechanism Of Midgut Digestive Juice Inhibiting BmNPV Infection In Silkworm Bombyx Mori Based On Label-Free

Bombyx mori is an important economic insect and a model organism of Lepidoptera insects.Bombyx mori nuclearpolyhedrosis virus(BmNPV)is one of the most serious pathogenic microorganisms in the sericulture industry,and it causes tremendous loss in sericulture every year.Infection of BmNPV in silkworm is mainly depend on oral infection in nature.B.mori has alkaline digestive juice and it triggers the dissolution of occlusion bodies(OBs)and the occlusion-derived virus(ODV)were release into the midgut lumen to infect the midgut.The silkworm digestive juice is the first element that interacts with BmNPV and it is probably the most important substance for silkworm resistance.In previous studies,some proteins were confirmed that they have the function of inactivating BmNPV in digestive juices.Therefore,studying the effect of midgut digestive juice on the invasion of BmNPV in silkworm is particularly important to analyze the resistance mechanism of silkworm.In this study,the silkworm susceptible strain P50 and resistant strain A35 were used as the experimental materials.Firstly,the differences in resistance between the digestive juices of A35 and P50 were analyzed,and the possible causes of the resistance differences were deduced.Further,based on the Label-free quantitative proteomics,the differentially expressed proteins(DEPs)were screened in P50 and A35 after BmNPV infection and the DEPs between P50 and A35,then candidate DEPs that may be involved in the BmNPV resistance of B.mori were successfully screened.Finally,the antiviral activity of two candidate DEPs(Lipase member H-A,BmLHA and Trypsin,alkaline A,BmTA)were analyzed.The major research results and conclusions are as follows:1.The analysis of resistant difference between different resistant strain P50 and A35 digestive juice.The virus copy number revealed that the virus proliferation in the intestine of P50 was significantly higher than that of A35 after BmNPV infection.The virus proliferation in the midgut of the control group was significantly higher than that in the two treatment groups which the silkworms were infected with OBs that were incubated with the digestive juice of the two strains,respectively.Additionally,the virus proliferation in the P50 digestive juice treatment group was significantly higher than that in A35 digestive juice treatment group.In addition,the p H value of the digestive juice of the two silkworm strains both gradually increased along with the fifth instar days increasing,but the values of p H showed non-significant difference between the two strains.2.Label-free-based quantitative proteomics analysis of the molecular mechanisms of Bombyx mori digestive juice in response to BmNPV in different resistant strains.Label-free analysis was conducted by using resistant silkworm strain A35 and susceptible silkworm strain P50.A total of 98 proteins were identified from four groups,including 68 enzyme proteins and 30 non-enzyme proteins.The 80 DEPs were screened in the four different comparison groups(P50 + vs P50-,A35 + vs A35-,A35 + vs P50 + and A35-vs P50-),including 29 serine proteases and 3 serine protease inhibitors.Utilizing Venn diagram analysis allowed for the removal of DEPs related to either genetic background(A35-vs P50-)and individual immune stress response proteins(P50+ vs P50-).Nine DEPs were identified potentially related to resistance to BmNPV.Additionally,based on previous research,the 16 proteins that are highly expressed in resistant strain as candidate DEPs that may be involved in BmNPV resistance in silkworm.3.Identification and functional analysis of B.mori Lipase member H-A(BmLHA).Based on proteomics data,we selected BmLHA from the candidate DEPs that may be involved in the resistance to BmNPV in silkworms for further research.The protein expression verification of BmLHA in the midgut digestive juice,the transcription level and protein level in the midgut were basically consistent with the proteomic data.Bioinformatics analysis indicated that BmLHA is a secreted protein and belongs to the lipase family.The tissue expression profiles of BmLHA indicated that it was strongly expressed in the midgut,and expressed at lower levels in other tissues.Additionally,the BmLHA only expressed in the larval stage,and the expression level significantly increased with the increasing of larve stage.Meanwhile,the different expression levels of BmLHA during moulting stage suggested that it may be regulated by hormones.The recombinant BmLHA incubation in vitro could reduce the infectivity of OBs and BV in silkworm larva and BmN cells.Moreover,over-expression of BmLHA in BmN cells could inhibit virus infection.These results confirmed that BmLHA is a digestive enzyme with BmNPV inhibitory activity.4.Identification and functional analysis of B.mori Trypsin,alkaline A(BmTA).Based on proteomics data,we selected BmTA from the candidate DEPs that may be involved in the resistance to BmNPV in silkworms for further research.The protein expression verification of BmTA in the midgut digestive juice,the transcription level and protein level in the midgut were completely consistent with the proteomic data.Bioinformatics analysis indicated that BmTA is a secreted protein and belongs to the serine protease family.The tissue expression profiles of BmTA indicated that it is specifically expressed in the midgut of the larva,and the expression level significantly increased with increasing of ingested leaves amount.Meanwhile,the different expression levels of BmTA during moulting stage suggest that it may be regulated by hormones.Recombinant BmTA could reduce the infectivity of OBs and BV in silkworm larve and BmN cells after incubation in vitro.Moreover,over-expression of BmTA in BmN cells could inhibit virus infection.These results confirmed that BmLHA is a digestive enzyme with BmNPV inhibitory activity.In summary,based on Label-free proteomics technology,the differences showed in digestive juice of different resistant strains in response to BmNPV were analyzed,and comprehensive and systematic analysis of the DEPs and their possible functions in response to BmNPV were also performed.Two candidate DEPs,BmLHA and BmTA,were confirmed that it has antiviral activity by various technical means.These results provide theoretical support for further analysis of resistance mechanisms of silkworm resistant strains in response to BmNPV infection.